The antitumor and antimicrobial functions of activated mouse macrophages are heavily dependent on the production of nitric oxide by the IFNgamma- and LPS-inducible enzyme, NO synthase (NOS). We call this isoform iNOS for its inducibility and independence of exogenous calmodulin (CaM) and elevated Ca2+. We have cloned iNOS and shown that its induction is transcriptional; beyond this, nothing is known of its regulation. Isoforms of NOS found in brain and endothelium differ from iNOS in two key respects. First, they are constitutive. Second, they are activated by transient association with exogenous CaM in the presence of elevated Ca2+ (hence termed cNOS). In contrast, we have shown that iNOS copurifies with CaM as a tightly bound subunit, even when Ca2+ is not elevated. NOS's similar to identical to macrophage iNOS are widely distributed in many cell types exposed to cytokines and LPS, such as liver, endothelium and vascular smooth muscle, where excessive NO production is believed to play a key role in septic shock. Anecdotal experience in man supports animal studies suggesting that inhibitors of NOS may prevent death from septic shock. However, other studies show that inhibition of cNOS can lead to platelet aggregation, coronary and cerebral ischemia, and death. iNOS-specific inhibitors are sorely needed. Their design could be aided by knowledge of the currently unknown binding sites for the substrate, L-arginine. iNOS appears to mediate a major portion of the antimicrobial and antitumor activity of the activated mouse macrophage. Very few studies have been able to identify a similar pathway in the human macrophage. It is baffling why an enzyme so important to defense of the mouse against intracellular infections of macrophages should be so hard to detect in human macrophages. We approach these three issues by focussing on macrophages derived from mouse peritoneum and human blood. A combined biochemical and molecular biologic approach will reveal how IFNgamma and LPS synergize to induce iNOS in the mouse, and will characterize the L-arginine and CaM binding sites through which enzyme activity is regulated physiologically and through which it might be inhibited pharmacologically. Finally, we will clone human iNOS, compare its expression in macrophages in vitro and in vivo, and clarify whether its apparent underexpression in human macrophages in vitro reflects culture conditions that favor deactivation; failure to employ physiologically optimal activating agents; use of assays for products of NOS whose reliability is compromised by alternative biochemical reactions; or a genetically regulated defect in the antimicrobial and antitumor repertoire of the human macrophage.